Clostridium perfringens epsilon toxin (ETX) is one of the most potent bacterial toxins, with a mouse lethal dose of 100ng/kg. The 32.5kD inactive prototoxin (E-PTX) is converted to the >1000-fold more toxic form by post secretion processing, and removal of a 22 amino acid C-terminal fragment is required for activation. While best known for its role in pathogenesis of enteritis and enterotoxemia in domestic animals, its high toxicity, ease of production, even in developing nations, and ability to act from mucosal surfaces make it a possible bioterror/biowarfare agent. Knowledge of ETX and its mode of action is limited, perhaps due to previous lack of a direct connection to human health issues. To expand our understanding of ETX, we propose the following specific aims; (1) To define and compare the crystal structure of ETX and E-PTX, and (2) to identify functional regions of ETX, through targeted mutagenesis. A prediction of the 3D structure of E-PTX is presented as preliminary data. In Aim 1, purified monodispersed protein will be used to grow suitable crystals of ETX for X ray diffraction and determination of 3D structure allowing comparisons which should reveal conformational changes associated with toxin activation. The preliminary structure of E-PTX indicates the presence of three structural domains, and it has been possible, based on this structure, to predict functional domains involved in receptor binding and membrane insertion of the toxin. In Aim 2, functional regions predicted from the crystal structure will be investigated by targeted mutagenesis. Domain 1, predicted to be involved in receptor binding, will be subjected to alanine scanning mutagenesis to define receptor-binding residues within this domain. Regions from domains 2 and 3, predicted to be involved in membrane insertion, will be analyzed by cysteine scanning mutagenesis and subsequent fluorescence based analysis of the environment of each amino acid in the presence of target cell membranes. Finally, regions involved in toxin oligomerization could not be predicted from structure of E-PTX, so a structure based approach will be undertaken in which surface exposed residues on the multiple faces of the ETX molecule will be mutated to determine which face is involved in oligomerization. These experiments will provide valuable information on the three important steps in ETX intoxication of target cells; cell binding, oligomerization and membrane insertion. This information can be used to model the mode of action of ETX, and to define regions of the molecule that could be targeted for immunoprophylaxis and treatment.